Search

SUMMARY Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root‐growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3‐BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8‐diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3‐BIO1 activity. Furthermore, cadaverine‐treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl‐coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants., Significance Statement Cadaverine is a polyamine produced by plants and microbes, which has been shown to accumulate in plants under stress conditions and to inhibit primary root growth. Here, we show that cadaverine affects Arabidopsis root growth primarily by inhibiting the biotin biosynthesis pathway, thereby affecting primary metabolism and the lipid profile.

SUMMARY The seed coat is specialized dead tissue protecting the plant embryo from mechanical and oxidative damage. Tannins, a type of flavonoids, are antioxidants known to accumulate in the Arabidopsis seed coat and transparent testa mutant seeds, deficient in flavonoid synthesis, exhibit low viability. However, their precise contribution to seed coat architecture and biophysics remains evasive. A seed coat cuticle, covering the endosperm outer surface and arising from the seed coat inner integument 1 cell layer was, intriguingly, previously shown to be more permeable in transparent testa mutants deficient not in cuticular component synthesis, but rather in flavonoid synthesis. Investigating the role of flavonoids in cuticle permeability led us to identify periclinal inner integument 1 tannic cell walls being attached, together with the cuticle, to the endosperm surface upon seed coat rupture. Hence, inner integument 1 tannic cell walls and the cuticle form two fused layers present at the surface of the exposed endosperm upon seed coat rupture, regulating its permeability. Their potential physiological role during seed germination is discussed., Significance Statement Tannins are seed coat antioxidants promoting seed viability and dormancy. A cuticle, covering the endosperm outer surface was previously shown to be more permeable in transparent testa mutants, which are deficient in flavonoid synthesis but not in cuticular component synthesis. We show that the endosperm outer surface is also covered by tannic cell walls arising from the seed coat inner integument 1 layer. Thus, tannic cell walls are endosperm outer surface structural components influencing its permeability.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases., PLoS Pathogens, 16 (6), ISSN:1553-7374, ISSN:1553-7366

Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae., Author Summary Plants control their flowering time in response to the temperatures of their environment, e.g. in response to the experience of winter or in response to cold and warm ambient temperatures experienced during spring. The knowledge about the evolutionary adaptation of plants to changing ambient temperatures is at present very limited. Understanding the latter is, however, becoming increasingly important due to the temperature changes associated with global warming and the anticipated changes in flowering time in ecosystems and agricultural systems. Here, we uncover an evolutionarily conserved molecular mechanism employed by Arabidopsis thaliana ecotypes for the adaptation of flowering time to cool temperatures. This structural change in the architecture of the gene FLOWERING LOCUS M can be found in multiple A. thaliana natural accessions and the knowledge gained in our study may be used to predict or modify flowering time in plants related to A. thaliana in the future.

As a consequence of a sessile lifestyle, plants are constantly facing a fluctuating environment. In order to both profit maximally and protect themselves from these environmental cues, plants evolved ways to sense and respond to signals. Ambient temperature is one of the cues for which plants have acquired a strategy to enhance their chance of survival and reproduction. Small changes in ambient temperature can have major effects on plant architecture and development, such as the transition from the vegetative to the reproductive flowering phase. The moment of flowering is an important event in the life cycle of a plant, since reproductive success depends on it. In Chapter 1, I introduced the concept of alternative splicing, a molecular mechanism with a pivotal role in ambient temperature regulation of flowering time. In the model plant Arabidopsis thaliana, approximately 60% of the intron-containing genes show alternative splicing. Gene splicing varies depending on developmental stage and tissue type, but also environmental changes trigger differential splicing. Splicing is conducted by a large cellular machinery called the spliceosome, which recognizes intron-defining sequences and other cis-regulatory elements acting as splicing enhancers or silencers. Moreover, factors like chromatin structure, histone marks, RNA polymerase II (polII) elongation speed and the secondary structure of the pre-mRNA all play a role in the splicing outcome. Due to alternative splicing, a single gene can yield various transcripts. However, this does not cause an equal expansion of the proteome. Part of the transcripts are targeted for nonsense-mediated decay, or will be translated into unstable proteins. This is a way of regulating gene expression at the post-transcriptional or –translational level. Other transcripts will be translated into functional proteins that may be structurally and functionally different. Hence, alternative splicing creates additional complexity in the transcriptome, providing plants with molecular tools to respond to their environment, including the translation of ambient temperature alterations into a flowering time response. In Chapter 2, we reviewed the current knowledge on molecular mechanisms that control the ambient-temperature directed flowering time pathway in the plant model species Arabidopsis thaliana. Several different mechanisms have been proposed, like alternative splicing of FLOWERING LOCUS M (FLM) (described in Chapter 4) and protein degradation of SHORT VEGETATIVE PHASE (SVP), two mechanisms that probably work in a cooperative manner to release floral repression at higher ambient temperatures. Another mechanism that is involved at high ambient temperature is the replacement of the canonical histone H2A by the variant H2A.Z. As a consequence of this replacement, chromatin becomes less tightly wrapped around the nucleosomes, which allows transcription of flowering time activators, such as PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Lastly, we discuss microRNAs (miRNA) that can either repress or activate flowering (miR156 and miR172, respectively). These miRNAs have been proposed to be regulated by low and high ambient temperature. However, due to the lack of mutant analyses, more research is necessary to show the true involvement of these factors. Altogether, there are several mechanisms acting partly in cooperation to regulate thermosensitive floral timing. In Chapter 3, we analysed ambient temperature-directed alternative splicing events that occur after a temperature shift by RNAseq. We performed the experiment in two different accessions of A. thaliana, and in one variant of B. oleracea (cauliflower). We showed that flowering time genes are overrepresented amongst the ambient temperature induced alternatively spliced genes, but also genes encoding components of the splicing machinery itself, indicating that alternative splicing is one of the potential mechanisms by which plants are able to sense temperature and adapt floral timing. Analysis of a mutant for one of these alternatively-spliced splicing related factors, ATU2AF65A, showed a temperature-dependent flowering time phenotype, confirming its proposed role in the flowering time response upon temperature fluctuations. Based on these findings, we proposed a two-step model in which splicing related genes are targeted for differential splicing upon ambient temperature fluctuations, which results in changes in the composition of the spliceosome, causing differential splicing of downstream genes that affect the development and architecture of the plant, including flowering time. In Chapter 4, we investigated the molecular mode-of-action of FLM, one of the differentially spliced flowering time regulating genes that we identified in Chapter 3. We showed that in A. thaliana Col-0, the main splice forms of FLM are FLMβ and FLMδ. FLMβ forms an obligate heterodimer with SVP, and this complex represses floral integrators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) by binding to the regulatory regions of these genes. FLMδ also dimerizes with SVP, but this complex is not able to bind to DNA. When temperature rises, more FLMδ is produced at the cost of FLMβ. Hence, less repressive complexes can be formed. However, the fact that FLMδ is still able to binds SVP makes it function as a dominant negative form, titrating out SVP and preventing repressive SVP/FLMβ-complex formation. Chapter 5 is a short comment written to clarify the concept of thermoplasticity in flowering time control. Occasionally, this concept is confused with adaptation to different ambient temperature environments on the long term. Thermoplasticity is the ability to adapt flowering time to fluctuations in ambient temperature within one life cycle. Furthermore, some genes have been marked as players in the ambient temperature response, whereas these appear to be general flowering repressors or activators, affecting flowering time in a similar manner at low and high ambient temperature. In order to interpret novel findings on thermosensitive flowering time control, it is essential to distinguish between these various concepts. In chapter 6, we unveiled the first indications that differential splicing of FLM can be caused by differences in polymerase II elongation rate. We mimicked a situation in which FLM is transcribed at a higher rate, by expressing the genomic FLM gene under a strong artificial promoter. Preliminary results showed that plants harbouring this construct have altered flowering time and temperature-responsiveness, which can be explained by the altered FLMβ/FLMδ ratio that we observed. In chapter 7, we assessed the functional conservation between FLM and closely related genes at the intraspecific level in A. thaliana. FLM (also called MAF1) is a member of the FLC-clade, that consists of FLC, FLM and MAF2-5. FLC is widely known for its function in the vernalization pathway, whereas MAF2 has been shown to regulate flowering time through alternative splicing in a way very similar to FLM. For the other MAF genes, not much is known. We showed that all of these genes produce splicing isoforms that function in a more or less similar way to FLM and MAF2. Despite the high functional conservation at the intraspecific level, FLM and MAF orthologues are not widely present. Through synteny analysis, we showed that FLM and MAF2 are very recent genes, which are only present in a small group of Brassicaceae species. MAF3-5 originated less recently, but are not present outside the Brassicaceae. For FLC, it was previously shown that it originated from an ancestor of the seed plants, and in many plant species belonging to other families, presence of more than one FLC-like gene has been reported. This raises the question what the function of these genes is. In tomato, we showed that the FLC-like gene MBP8 becomes differentially spliced upon temperature changes, suggesting a function in the ambient temperature pathway. A binding assay showed high similarities of the different MBP8 isoforms to FLM and MAF isoforms, but suggests a slightly different functionality, since all three isoforms showed binding to the DNA. Further research is necessary to confirm the role of MBP8 in thermosensitive flowering time control, and elucidate the functionality of the different splice forms. In Chapter 8, I discussed the finding of this thesis in a broader perspective, and make suggestions for future research. Over the last few years, several mechanisms that act in the temperature-directed floral pathway have been revealed. In this thesis, we showed that alternative splicing plays an important role, and we demonstrated how temperature may affect the splicing outcome directly through the effect of temperature on transcription elongation rate. It is becoming clear that most likely a single thermosensor does not exist in plants, and a model in which temperature is sensed through thermodynamic properties of DNA, RNA and proteins, is gaining support. Future research is assigned to the exiting task to elucidate the exact mechanisms by which temperature-sensing is achieved in different plant species and to determine how conserved the currently identified molecular mechanisms are.

The floral transition is an essential process in the life cycle of flower-bearing plants, because their reproductive success depends on it. To determine the right moment of flowering, plants respond to many environmental signals, including day length, light quality, and temperature. Small changes in ambient temperature also affect the flowering process, although our knowledge of the genetic and molecular mechanisms underlying this flowering pathway is limited. However, recent advances in Arabidopsis (Arabidopsis thaliana) have uncovered multiple molecular mechanisms controlling ambient temperature regulation of flowering, which modulate both repressing and activating factors of flowering time. At a time when temperatures are rising worldwide, understanding how plants integrate ambient temperature signals can be crucial for crop production.

The appropriate timing of flowering is crucial for plant reproductive success. It is therefore not surprising that intricate genetic networks have evolved to perceive and integrate both endogenous and environmental signals, such as carbohydrate and hormonal status, photoperiod and temperature. In contrast to our detailed understanding of the vernalization pathway, little is known about how flowering time is controlled in response to changes in the ambient growth temperature. In Arabidopsis thaliana, the MADS-box transcription factor genes FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) have key roles in this process. FLM is subject to temperature-dependent alternative splicing. Here we report that the two main FLM protein splice variants, FLM-β and FLM-δ, compete for interaction with the floral repressor SVP. The SVP-FLM-β complex is predominately formed at low temperatures and prevents precocious flowering. By contrast, the competing SVP-FLM-δ complex is impaired in DNA binding and acts as a dominant-negative activator of flowering at higher temperatures. Our results show a new mechanism that controls the timing of the floral transition in response to changes in ambient temperature. A better understanding of how temperature controls the molecular mechanisms of flowering will be important to cope with current changes in global climate.